Abstract
Background/Aim: Glioblastoma multiforme is the most malignant type of glioma. Ecto-5’-nucleotidase (ecto-5’NT), a glioma-overexpressed enzyme can induce a protective effect on tumor cells. Monastrol, a kinesin spindle protein-specific inhibitor, is reported to be an interesting prototype for cancer therapy. We describe the effect of LaSOM 63, a monastrol derivative, on ecto-5’NT activity and on glioma cell survival. Materials and Methods: Glioma cells were treated with LaSOM 63 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypan blue assay (viability), flow cytometry (cell cycle/cell death) and malachite green method for ecto-5’NT activity were carried out. Results and Discussion: Treatment with LaSOM 63 reduces glioma cell viability and cell growth. In contrast to monastrol, LaSOM 63 did not cause glioma cell-cycle arrest, but inhibited ecto-5’NT enzyme activity. Furthermore, this compound induces apoptotic death of C6 and U138 glioma cells. Conclusion: LaSOM 63 may be useful for in vivo experiments on the treatment of GBM.
Gliomas are the most common brain tumors. Among them, glioblastoma multiforme (GBM, referred to as grade IV by the World Health Organization) is the deadliest type (1). The infiltrative feature of these cancer cells throughout the brain is the main reason for failure of GBM resection. Moreover, remaining cancer cells are able to self-renew, even when in small number, the creating a new tumor (2). Survival rates have not increased over the past years due to robust cell proliferation, neo-angiogenesis, immunosuppression and intrinsic resistance to radiation and chemotherapy presented by this tumor type (3). Despite the great efforts made over the past decades, few GBM patients survive more than five years (4).
The 4-aryl-3,4-dihydropyrimidin-2(1H)-ones (or simply dihydropyrimidinones) are a class of heterocyclic compounds usually obtained through Biginelli reaction that has monastrol as their prototype. This molecule exerts specific inhibition over Eg5, a mitotic kinesin that is required for bipolar spindle formation, leading to cell-cycle arrest in the mitotic phase (5, 6). This inhibition is considered an attractive approach to cancer treatment since kinesins are expressed only in proliferating cells and do not cause neurotoxicity as classic mitotic inhibitors do (7). Monastrol has antitumor activity against diverse cancer cell types such as renal, breast and glioma cell lines (8, 9). Thus, efforts towards developing monastrol-like structures are currently increasing.
ATP, ADP, and AMP hydrolysis are finely-controlled by membrane bound ecto-enzymes in the extracellular medium. The final step of the hydrolysis cascade is mediated by ecto-5’-nucleotidase/CD73 (ecto-5’NT), which hydrolyzes nucleoside monophosphates such as AMP to adenosine [for a complete review see Zimmermann et al. 2012 (10)]. Ecto-5’NT has been found to be overexpressed in several types of cancers (11). Ecto-5’NT activity is important for GBM proliferation and is increased in these cells when compared to astrocytes (12). In this context, the decrease in cell proliferation with α,β-methylene ADP (APCP) treatment, a synthetic inhibitor of this enzyme, as well as AMP cytotoxicity on GBM cell lines, corroborate with the importance of ecto-5’NT and adenosine in tumor promotion (13).
Considering the high malignancy and poor prognosis of GBM, the interest in and necessity for developing new treatments, that are more effective and safe, are increasing. With this purpose in mind, we have synthesized LaSOM 63 [5-ethoxycarbonyl-6-methyl-4-(4-N,N-dimethylaminophenyl)-3,4-dihydropyrimidin-2-(1H)-thione] and monastrol [5-ethoxycarbonyl-6-methyl-4-(3-hydroxyphenyl)-3,4-dihydropyrimidin-2-(1H)-thione] (Figure 1), as previously reported, using an environment-friendly methodology based on the combined use of citric or oxalic acid and triethylorthoformate via Biginelli multicomponent reaction (9). The antiproliferative effect of LaSOM 63 on glioblastoma cell lines and the mechanism by which this molecule exerts its effects was evaluated in the present study.
Materials and Methods
Maintenance of cell lines. The rat C6 and human U138 glioma cell lines were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA). Cells were grown and maintained in 1% Dulbecco's modified Eagle's medium (DMEM) containing antibiotics (0.5 U/ml penicillin/streptomycin) and supplemented with 5% and 10% (v/v) fetal bovine serum (FBS), respectively (all from Gibco BRL, Carlsbad, CA, USA). Cells were kept at a temperature of 37°C, minimum relative humidity of 95%, and atmosphere of 5% CO2 in air.
Assessment of glioma cell viability. For the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, C6 glioma cells were seeded in 96-well plates and allowed to grow until semi-confluent. Cells were treated with 10, 25, 50, 75, 100 and 200 μM of LaSOM 63 for 48 h. At the end of the treatment, MTT (5 mg/ml) was added to each well. A total of 100 μl dimethyl sulfoxide (DMSO) was added to the wells and the level of absorbance was read at 570-630 nm. For the trypan blue (Sigma, St. Louis, MO, USA) dye exclusion test, C6 glioma cells were seeded in 24-well plates and allowed to grow until semi-confluent. Treatment was carried out for 48 h at the concentrations of 25, 50 and 100 μM of LaSOM 63. At the end of the treatment, 100 μl of 0.05% trypsin/EDTA (Gibco BRL) solution was added to detach the cells, which were counted immediately in a hemocytometer with trypan blue. In all experiments, statistical analysis was performed comparing data to that for vehicle control groups treated with DMSO.
Cell-cycle analysis. Cells were plated in 6-well plates, and after reaching semi-confluence they were treated with 100 μM LaSOM 63 or 100 μM monastrol (positive control) for 24 h. At the end of treatment, the cell medium was removed and cells were washed twice with PBS (pH 7.4), harvested, centrifuged, and suspended with 400 μL staining solution [Tris-HCl 0.5 mM (pH 7.6); 3.5 mM trisodium citrate; 0.1% (v/v) NP40; 100 μg/mL RNAse; 50 μg/ml propidium iodide (PI)] at a density of 106 cells/ml. After 30 min, data were collected using a flow cytometer (FACS Calibur cytometric system; BD Bioscience, Mountain View, CA, USA) and analyzed by FLOWJO® software.
Ecto-5’NT activity. Ecto-5’NT activity was assayed as described by Wink et al. (12). 24-Well plates containing glioma cells that were exposed to LaSOM 63 for 48 h were washed three times with phosphate-free incubation medium. Enzymatic reaction was started by the addition of 200 μl of incubation medium containing 2 mM AMP at 37°C. After 10 min of incubation, the reaction was stopped when 150 μl of the incubation medium in contact with the cells was collected and transferred to an eppendorf tube containing trichloroacetic acid (10% w/v) previously placed on ice. Inorganic phosphate released was measured by the malachite green method (14). Control samples were performed to determine non-enzymatic inorganic phosphate release. Protein was quantified by the Comassie blue method (15) utilizing bovine serum albumin as standard. Specific activity was expressed as nanomoles of inorganic phosphate released/min/mg of protein.
Molecular structure of monastrol and LaSOM 63.
Annexin V/PI assay. Apoptotic cells were quantified using an AnnexinV-fluorescein isothiocynate-PI (AnnexinV-FITC–PI) double staining kit, according to the manufacturer's instructions (BD Biosciences, San Diego, CA, USA). C6 rat and U138 human glioma cells were plated in 6-well plates and treated with 50, 100 and 200 μM LaSOM 63 for 48 h. At the end of the treatment, cells were washed twice with cold PBS (pH 7.4) and counted. Next, 105 cells were suspended in binding buffer containing FITC-conjugated annexinV and PI. Samples were then agitated and incubated for 15 min at room temperature in the dark. Apoptotic and necrotic cells were quantified using a dual-color flow cytometric technique on a FACS Calibur cytometric system (FACS Calibur; BD Bioscience, Mountain View, CA, USA). Data obtained were analyzed by the FLOWJO® software (Tree Star, INC Ashland, OR, USA). Cells were classified as follows: live, annexin−/PI−; early apoptotic, annexin+/PI−; late apoptotic, annexin+/PI+, and necrotic, annexin−/PI+.
Statistical analysis. Data were analyzed for statistical significance by one-way analysis of variance (ANOVA) followed by a post-hoc test for multiple comparisons (Tukey test) using GraphPad Prism Software® (GraphPad Software, INC, La Jolla, CA, USA). Data are expressed as the mean±S.E.M. Differences were considered significant at p<0.05.
Results
LaSOM 63 reduced C6 glioma cell viability. To investigate the potential cytotoxicity of LaSOM 63 on glioma cells, the MTT and trypan blue dye exclusion assays were used. C6-cultured cells were treated for 48 h as described in the Materials and Methods. Analysis by the MTT assay showed that 75, 100 and 200 μM LaSOM 63 caused a significant reduction in cell viability compared to DMSO-treated cells (25%, 28% and 55%, respectively) (Figure 2A). In agreement with this, trypan blue exclusion assay showed a significant decrease in cell growth at 50 and 100 μM LaSOM 63 compared to the DMSO-treated cells (40% and 52%, respectively) (Figure 2B). DMSO-alone did not cause significant alteration on glioma C6 cell viability in the two used tests.
LaSOM 63 reduces viability and growth of C6 glioma cells. C6 glioma cells were treated for 48 h with LaSOM 63 (10 to 200 μM, MTT assay) and (25, 50 and 100 μM, trypan blue exclusion assay). The percentage of cell viability was calculated in relation to control cells, considered as 100% viability. Data presented are the means±S.E.M. of three independent experiments. Data were analyzed by one-way ANOVA followed by post-hoc comparisons (Tukey). *Significantly different from the DMSO-treated cells (p<0.05).
LaSOM 63 does not alter cell-cycle progression. C6 glioma cells were treated with DMSO, 100 μM LaSOM 63 (63) or 100 μM monastrol (M) for 24 h. After treatment, cells were permeabilized and stained with propidium iodide for determination of cell-cycle distribution (sub-G1, G0/G1, S and G2/M) by flow cytometry. Peak areas in the histogram were calculated using FLOWJO® software. A: Histogram representation. B: Data are presented as the means±S.E.M. and were analyzed by one-way ANOVA followed by post-hoc comparisons (Tukey). *Significantly different from the DMSO-treated group (p<0.05).
LaSOM 63 inhibits ecto-5’NT/CD73 activity in C6 glioma cell line. C6 glioma cells were treated with 10, 25, 50, 75, 100 and 200 μM of LaSOM 63 for 48 h. After treatment, cells were submitted to an enzymatic assay for AMP hydrolysis (release of inorganic phosphate, Pi) by malachite green method. Specific activity of ecto-5’NT is expressed as nmol Pi/min/mg protein. Data represent the mean±S.E.M. of four independent experiments. Data were analyzed by one-way ANOVA followed by post-hoc comparisons (Tukey). *Significantly different from the DMSO-treated group (p<0.05).
LaSOM 63 did not alter cell-cycle progression. Considering the well-established effect of monastrol causing cell-cycle arrest in the G2/M phase, we tested the effect of LaSOM 63, on cell-cycle progression (Figure 3). Cell-cycle analysis showed that unlike monastrol, LaSOM 63 did not alter cell-cycle progression (G2/M phase for DMSO=9.4%; LaSOM 63=4.5%; monastrol=80.0%). However, LaSOM 63 increased the sub-G1 phase proportion, which indicates apoptotic death of C6 glioma cells (Figure 3A and B).
LaSOM 63 inhibited ecto-5’-nucleotidase activity in C6 glioma cells. As LaSOM 63 and monastrol have different behaviors in glioma cell-cycle progression, we investigated the effect of LaSOM 63 on ecto-5’NT activity, since monastrol did not alter activity of this enzyme (unpublished results). C6 glioma cells were treated with LaSOM 63 and after treatment, inorganic phosphate release from AMP hydrolysis was measured as described in Material and Methods. Treatment with LaSOM 63 inhibited ecto-5’NT activity at 100 and 200 μM (36% and 48%, respectively) (Figure 4).
LaSOM 63 treatment induced apoptotic cell death of rat C6 and human U138 glioma cells. To better-evaluate the sub-G1 increase we next investigated whether LaSOM 63 induced apoptotic cell death. Flow cytometric analysis showed that LaSOM 63 did not increase PI incorporation into C6 and U138 cells, indicating the absence of necrotic cell death. Similar results were obtained for late apoptosis, where no Annexin-FITC+/PI+-stained cells were observed (both cell lines; data not shown). However, treatment with LaSOM 63 promoted a significant increase in the number of Annexin-FITC+/PI− cells compared to DMSO, suggesting early apoptotic cell death at 100 and 200 μM of LaSOM 63 (11.4% and 16.3%, respectively) for C6 cells and at 200 μM (24.03%) for U138 cells (Figure 5).
LaSOM 63 induces early apoptotic death of C6 and U138 glioma cells. C6 and U138 glioma cells were treated with 50, 100 and 200 μM of LaSOM 63 for 48 h. Apoptosis was measured by flow cytometry with Annexin V/propidium iodide staining. Data were analyzed by the FLOWJO® software. Data are representative of cells in early apoptosis only. The values presented are the means±S.E.M. and were analyzed by one-way ANOVA followed by post-hoc comparisons (Tukey). *Significantly different from the DMSO (D)-treated group to C6 cells (p<0.05). #Significantly different from the DMSO (D)-treated group, to U138 cells (p<0.05).
Discussion
The heterogeneity of GBM provides a challenge in the search for new therapies that target different signaling pathways. The diverse biological activity exhibited by Biginelli adducts recently brought new perspectives to the use of dihydropyrimidones in cancer research (16). Herein, we described that LaSOM 63, a monastrol derivative (Figure 1), exerts cytotoxic activity against glioblastoma cell lines. First, we showed a decrease in C6 cell viability based on the mitochondrial reduction of tetrazolium bromide salt to its formazan product (Figure 2A). We then confirmed by trypan blue dye exclusion assay that LaSOM 63 induces significant growth inhibition after treatment at 50 μM and 100 μM for 48 h (Figure 2B).
The effect of monastrol adducts on cancer cells has been consistently shown since this molecule was first-described in 1999 (5). Recently, we demonstrated that some monastrol analogs, obtained via Biginelli reaction using a new environment-friendly methodology, reduce GBM cell viability (9, 17). In other work, we have also shown that LaSOM 65, a monastrol derived-compound with a nitrophenyl substituent, did not arrest mitosis in glioma cells as monastrol does (15). Furthermore, this monastrol adduct has pharmacokinetic feasibility and is safe on acute treatment (18). Studies have described that monastrol and some derivatives exert antiproliferative/cytotoxic activity against tumor cells through a variety of mechanisms, depending on the cell origin, and this is associated with nitrogen/oxygen scavengers (19). These different signaling pathways have also been studied by others; Groth-Pedersen and collaborators showed that monastrol induced lysosomal membrane permeabilization and sensitized different cancer cell lines to other drugs (20).
Monastrol affects the mitotic machinery by inhibiting the kinesin Eg5-associated with tubulin protein arresting cell-cycle progression in G2/M phase (5); this has been confirmed by the results presented herein (Figure 3). On the other hand, LaSOM 63, which has a dimethylamine group in the “para” position instead of the hydroxyl group in the “meta” position of the aromatic ring in relation to monastrol, did not alter cell cycle progression (Figure 3). Monastrol can cause mitotic arrest by activating the spindle assembly checkpoint. However, attenuation of the spindle checkpoint can confer resistance to Kinesin Spindle Protein (KSP) inhibitor (21).
LaSOM 63 did not alter KSP function (shown by mitotic progression of C6 GBM cells, Figure 3), but can induce apoptosis of GBM cells by a different mechanism. Ecto-5’NT is an important enzyme which controls the adenosine concentration in the extracellular medium. Herein, we showed that LaSOM 63 reduces ecto-5’NT activity and consequently adenosine production in C6 glioma cells (Figure 4). C6 cell apoptosis can be mediated, at least in part, by a decrease in extracellular adenosine, since adenosine can stimulate glioma cell proliferation (22) and prevent drug-induced apoptosis of cancer cells (21). It is also possible that cell death can be induced by an increase in the concentration of AMP mediated by ecto-5’NT inhibition, as was previously shown (13).
As mentioned above, LaSOM 63 reduces GBM cell viability (Figure 2) without any antiproliferative effect (Figure 3). Thus, to investigate if LaSOM 63 induces a cytotoxic effect on GBM cells, we stained the cells with annexin V/PI. Figure 5 shows an increase in the number of apoptotic cells after treatment for 48 h with LaSOM 63. Apoptosis was also confirmed in the human GBM cell line U138-MG, which reinforces the results seen in the rat cell line (C6) (sub-G1 phase increase (Figure 3) and from annexin-V staining (Figure 5), supporting the hypothesis of apoptotic death. Prolonged exposure to KSP enzyme inhibitors can induce cell apoptosis by activating BCL2 associated X protein (Bax), a pro-apoptotic protein (21) or arrest in cell-cycle progression with caspase activation and loss of mitochondrial membrane potential (24). Nevertheless, we cannot exclude other forms of GBM cell death induced by monastrol adducts. For example, mitotic catastrophe caused by Eg5 inhibitors has also been reported (25).
In spite of advances in therapeutics, GBM remains a lethal type of cancer. Efforts should be made not only to find effective drugs, but also to develop alternative treatments that do not significantly affect the patient's quality of life. Improvements over older therapies that interfere with mitotic process, such as taxanes or vinca alkaloids that bind to tubulin are necessary to reduce side-effects in patients. LaSOM 63 appears to be a promising molecule for such proposes and although a monastrol analog, it acts through a different pathway. However, more studies are necessary to confirm its pre-clinical feasibility.
Acknowledgements
This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), and the Instituto Nacional de Ciência e Tecnologia para Inovação Farmacêutica (INCT-IF/CNPq).
- Received October 3, 2013.
- Revision received November 9, 2013.
- Accepted December 13, 2013.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
